The nervous system is derived from multipotential precursor cells that show a closely regulated inverse relationship between cell proliferation and differentiation (Cattaneo and McKay (1991) TINS 14: 338-340). In the central nervous system, these precursor cells commit to a specific differentiation pathway shortly after the last mitosis (McConnell (1988) J. Neurosci. 8: 945-974; Turner and Cepko (1987) Nature 328: 131-136). In the peripheral nervous system, sensory neurons differentiate following withdrawal from the cell cycle (Rohrer and Thoenen (1987) J. Neurosci. 7: 3739-3748), but sympathetic neurons begin to differentiate, expressing neurotransmitter systems and extending short neurites, while still mitotically active (DiCicco-Bloom et al. (1990) J. Cell Bio. 110: 2073-2086). The molecular basis of the coupling between neuronal differentiation and cell proliferation is a problem of current interest.
The coupling between neuronal differentiation and cell proliferation also is relevant to the etiology of neural tumors, such as neuroblastoma. Neuroblastoma is one of the most common pediatric solid tumors, frequently occurring in infancy with the primary lesion in the adrenals and sympathetic chain (Voute (1984) "Neuroblastoma in: Clinical Pediatric Oncology" (W. W. Sutow, D. J. Fernback and T. J. Vietti, ed.) pp 559-587). This tumor is difficult to treat as common modes of chemotherapy have harsh side effects on normal infant tissue. Interestingly, neuroblastomas are noted for their ability to undergo spontaneous regression or maturation to benign ganglioneuromas (Evans et al. (1980) Cancer 45: 833-839). The similarity of neuroblastoma cells to neuroblasts and their ability to spontaneously mature to a more benign form suggest that the disease may originate by a block of differentiation of a sympathetic precursor cell (Knudson and Meadows (1980) New Engl. J. Med. 302: 1254-1256). Hence, factors which promote the differentiation of proliferating neuroblastic cells are candidates for new therapeutic approaches. Due to the side effects of cancer therapy, there is great need for "natural" but highly-specific pharmaceutical treatments.
Nerve growth factor (NGF) is a 26,000-dalton polypeptide neurotrophic factor that mediates a variety of biological responses (Levi-Montalcini and Aloe (1987) Science 237: 1154-1162). NGF acts as a survival factor for sympathetic and sensory neurons both in vivo and in culture (Johnson et al. (1986) Trends Neurosci. 9: 33-37). NGF is a differentiation factor for pheochromocytoma cell line PC12 (Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73: 2424-2428). The effects of NGF on cell division vary with cell type. PC12 cells nearly cease to divide after exposure to NGF (Greene and Tischler (1976) Proc. Natl. Acad. Sci. USA 73: 2424-2428). Fetal chromaffin cells (Lillien and Claude (1985) Nature 317: 632-634) divide several times in response to NGF before terminally differentiating. Neuronal precursor cells from embryonic striatum proliferate in response to NGF but only after exposure to basic fibroblast growth factor (Cattaneo and McKay (1990) Nature 347: 762-765).
The use of cell differentiation factors such as NGF alone, however, has proven insufficient for the effective treatment of neuroblastomas. First, the response of neuroblastoma cell lines to NGF varies (Azar et al. (1990) Cell Growth Diff. 1: 421-428; Chen et al. (1990) Cell Growth Diff. 1: 79-85; Sonnenfeld and Ishii (1982) J. Neurosci. Res. 8: 375-391). Neuroblastoma cell lines with amplified N-myc oncogene have little or no response to NGF. SHSY5Y and some other neuroblastoma cell lines with single-copy N-myc extend short, branched neurites in response to NGF (Chen et al. (1990) Cell Growth Diff. 1: 79-85). However, NGF does not slow the rate of proliferation of these cells, and the differentiation is reversible. Upon the removal of NGF, the neurites retract. The inability of neuroblastoma cells to terminally differentiate may be a critical factor in this disease (Azar et al. (1990) Cell Growth Diff. 1: 421-428). As described in the references discussed below, blockade of cell division, alone, does not appear to be sufficient to promote fiber outgrowth. On the other hand, the compound aphidicolin has been reported to enhance the differentiation of human cell lines. Aphidicolin is a steroid-like molecule isolated from fungi which reversibly inhibits DNA polymerase .alpha. and .delta. and blocks the cell cycle at G.sub.1 /S (Huberman (1981) Cell 23: 647-648). Chou and Chervenick (Cell Tissue Kinet. (1985) 18: 387-397) reported that a low concentration (0.4 .mu.M) of aphidicolin enhances the retinoic acid-induced differentiation of human leukemia cells. Jensen (Dev. Biol. (1987) 120: 56-64) found that SHSY5Y cells treated with NGF and a pulse of aphidicolin extend long neurites (greater than 300 .mu.m) and irreversibly differentiate. A high, and lethal, concentration (30 .mu.M) of aphidicolin was used by Jensen to select postmitotic SHSY5Y cells, resulting in considerable cell toxicity. Combining NGF and sublethal doses of aphidicolin to effect the growth of SHSY5Y cells and still provide beneficial results was not discussed until the present invention.
For example, Griffin et al. (Exp. Haematology (1982) 10: 774-781) monitored the effects of two specific inhibitors of DNA synthesis, cytosine arabinoside and aphidicolin, to determine whether the slowing of DNA polymerization can induce differentiation in HL-60 human leukemic promyelocytes. The results indicated that cytosine arabinoside and aphidicolin both induced cellular differentiation in HL-60 cells. The inhibition of DNA synthesis, and thereby of cellular replication, may permit cells to express genetic information that results in a differentiated phenotype. The normal balance between proliferation and differentiation which has been lost in myeloblastic leukemic cells may be partially restored by blocking proliferation with drugs such as cytosine arabinoside or aphidicolin. No recommendation as to specific methods to accomplish this goal for different cell lines is provided.
Sonnenfeld and Ishii (J. Neurosci. Res. (1982) 8: 375-391) investigated whether the response of cultured human neuroblastoma cells to NGF was altered in a manner consistent with the pattern of decreased sensitivity to normal growth regulators exhibited in other malignant transformed cell types. A number of cell lines were examined to assess the degree of variability in response between cell lines. Thus an altered response to NGF may be associated with human neuroblastoma. It is pointed out that regulation of neurite outgrowth and cellular growth or proliferation are separable in neuroblastoma. However, Sonnenfeld and Ishii failed to demonstrate that NGF reduced the growth rate or survival of any neuroblastoma cell line.
Chou and Chervenick (Cell Tissue Kinetics (1985) 18: 387-397) evaluated the relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukemic (HL-60) cells with the use of aphidicolin. The addition of a sublethal concentration of aphidicolin (0.4 .mu.M) in culture for three days suppressed DNA synthesis to a similar level of the resting stage in control cultures. DNA synthesis and cell proliferation was reactivated to the level observed in the growing stage of control cultures once aphidicolin was removed after three days in culture. The inhibitory effect of aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures appeared to be similar. However, no reactivation of DNA synthesis was observed after removal of aphidicolin on day 3 from RA-induced cell cultures. It was pointed out that cells accumulated in G.sub.1 and early S phases of the cell cycle after exposure to aphidicolin with or without RA. Aphidicolin alone did not induce cells to differentiate. The rate of RA-induced cell differentiation in the presence of aphidicolin was similar to that of RA treated cultures in the absence of aphidicolin. It was suggested that the combined use of aphidicolin and RA may effectively inhibit leukemic cell proliferation without causing severe cytotoxicity and without interfering with RA induced cell differentiation. The viability of HL-60 cells was assessed through exposure to 0.2 .mu.M retinoic acid and/or 0.4 .mu.M aphidicolin in RPMI-FCS medium. Aphidicolin, when present, was removed from cells by washing with medium at day 3 after seeding. It was determined that treatment of leukaemic cells with aphidicolin for a period of one doubling of the cell numbers suppresses DNA replication without influencing RA-induced cell differentiation. The authors make no suggestion that aphidicolin may act together with a neurotrophic factor to enhance the differentiation potential of a neurotrophic factor.
Packard (Proc. Natl. Acad. Sci. USA (1987) 84: 9015-9019)reports that a synthetic nonapeptide fragment of thrombin inhibits the cellular motility in culture of a human melanoma subclone that possesses a high metatastic potential in mice. Pre-treatment of cells with this nonapeptide did not block signal transduction through plasma membrane receptors for the following growth or differentiation factors: .alpha.-melanotropin, NGF, and transforming growth factor type .beta..
Jensen (Developmental Biol. (1987) 120: 56-64), examined the potential of human neuroblastoma cell line SHSY5Y to differentiate in vitro after prolonged exposure to 7S NGF. SHSY5Y cells exposed to 7S NGF for periods exceeding five weeks and selected with aphidicolin closely resembled mature neurons as judged by several criteria. The treated and selected neurons survived for prolonged periods in culture in the presence of NGF. Human neuroblastoma SHSY5Y cultures were exposed to murine 7S NGF for five weeks and subsequently selected with aphidicolin for one week those cells were no longer mitotically active. It was pointed out that aphidicolin is a reversible inhibitor of .alpha.DNA polymerase and therefore kills mitotically active cells with prolonged exposure.
Thus, aphidicolin was utilized by Jensen simply to kill mitotically active cells. The addition of aphidicolin during the second week of culture treatment is necessary as even a small population of mitotically active cells will quickly overgrow a mitotically quiescent culture. The aphidicolin selection step was only introduced to compensate for apparent variabilities in the timing of differentiation and for the small degree of phenotypic and/or genetic instability which appeared to exist within the undifferentiated SHSY5Y cells. Aphidicolin was not added to enhance differentiation.
Moreover, Jensen fails to mention, as disclosed by the present invention, that a sublethal dose of aphidicolin enhances the capacity of NGF to promote differentiation of neuroblastoma cells. Instead, Jensen used a high, lethal concentration (30 .mu.M) to kill mitotically active cells.
Additionally, aphidicolin does not promote morphological changes in the absence of NGF. Jensen does point out that aphidicolin causes HeLa cells to accumulate in the G.sub.1 stage of the cell cycle and suggests that aphidicolin thus may act synergistically with NGF to promote entry into G.sub.0. Under similar conditions, retinoic acid also promoted certain aspects of a differentiated phenotype. Retinoic acidaphidicolin treated cultures exhibited similar morphological differentiation to the NGF treated cultures.
Although Jensen subjected neuroblastoma to a combination of NGF and aphidicolin, the dose of aphidicolin used and the time the aphidicolin was applied to the cells is distinct from the novel method. Furthermore, Jensen indicates that aphidicolin alone does not induce differentiation. Although there is a suggestion that aphidicolin may act synergistically with NGF to promote entry into G.sub.0, there is no relation of entry into G.sub.0 to differentiation. Moreover, Jensen does not mention that a sublethal dose of aphidicolin may enhance the capacity of NGF to promote differentiation of neuroblastoma cells.
Goretzki et al. (Surgery (1987) 102: 1035-1042) investigated whether sensitivity of human medullary thyroid carcinoma (hMTC) cells to chemotherapeutic drugs could be increased in vitro to initiate a more effective adjuvant chemotherapeutic approach for patients who undergo only palliative surgery. They report that NGF stimulated .sup.3 H-thymidine incorporation into hMTC cells according to dose and caused an enhanced cell proliferation in these cells up to threefold. Pre-incubation with NGF for 24 hours stimulated hMTC cells and made them more sensitive to cytotoxic therapy with doxorubicin. Stimulation of proliferation, i.e., hMTC and other APUD cells with NGF, enhanced the cytotoxicity of chemotherapeutic drugs to these cells. Therefore, Goretzki appears to teach away from the combination of a cytostatic or cytotoxic compound with NGF to enhance differentiation of cancer cells.
Cattaneo and McKay (Nature (1990) 347: 762-765) report that NGF controls the proliferation of neuronal precursors in a defined culture system of cells derived from the early embryonic brain. These cells proliferated in response to NGF, but only after they had been exposed to basic fibroblast growth factor. On withdrawal of NGF, the proliferative cells differentiated into neurons. It was indicated that, in combination with other growth factors, NGF regulates the proliferation and terminal differentiation of neuroethothelial cells. The authors suggest that NGF and other members of the NGF family might promote both the proliferation of neuronal precursors and the survival/differentiation of neurons derived from these precursors.
Chen et al. (Cell Growth and Differentiation (1990) 1: 79-85) examined a series of neuroblastoma and neuroepithelioma cell lines for NGF-induced neurite extension and NGF modulation of the expression of neuronal markers. The results indicated that three neuroblastoma cell lines with a neuronal morphology and lacking N-myc amplification extended neurites in response to 200 ng/ml of NGF. The authors conclude that NGF-induced differentiation is confined to a particular class of neural-related tumors, and, furthermore, differentiation for these cell lines is incomplete.
Unlike the articles discussed above, in the present invention, NGF and a pulse of aphidicolin were used at sublethal concentrations to induce efficient differentiation of SHSY5Y cells with little resulting toxicity. Under these conditions, the neuroblastoma cells cease to proliferate and instead extend long neurites. This is the first demonstration of such unexpected and synergistic effects of a neurotrophic factor and a cell cycle blocker. This invention provides a model system for the study of the coupling of cell proliferation and neuronal differentiation, as well as a novel method of treating neuroblastomas and other tumors.